The present invention relates to the use of cell specific vectors in the diagnosis as well as the prophylactic and therapeutic treatment of diseases of the breast, in particular cancer.
Cancers of the breast are one of the leading causes of death among women, with the cumulative lifetime risk of a woman developing breast cancer estimated to be 1 in 9. Consequently, understanding the origins of these malignancies as well as the identification of new therapeutic modalities is of significant interest to health care professionals.
The mature human breast comprises from six to nine major ducts, which emanate from the nipple, serially branch into ducts and terminate in lobuloalveolar structures (Russo et al., Lab. Invest. 62(3): 244-278 (1990)). The branching network of ducts is composed of luminal epithelial cells in a supporting matrix of connective tissue and myoepithelial cells. Tissues removed from the human female breast during surgery and autopsy have been examined in numerous studies directed at the nature and site of origin of neoplastic growth. Subgross sampling and histological confirmation have enabled pathological characterization of entire breasts which shows that human breast cancer arise within the ductal system of the breast exclusively from luminal epithelial cells. Ductal origin is supported by the presence of more extensive epithelial proliferations, which are presumed to be preneoplastic, in surgically removed cancerous breasts as compared to nonmalignant breasts removed during autopsies.
With the significant cumulative lifetime risk of a woman developing breast cancer, there is an urgent need to develop both therapeutic methods of treatment that are more effective, less invasive and accompanied by fewer side effects as well as prophylactic methods of treatment that are more effective than increased and intensified physical monitoring and far less extreme than radical mastectomy. In spite of the recent discovery of the heritable breast cancer susceptibility loci including BRCA1 and other genes (see e.g. Miki et al., Science 266:66-71 (1994)), and the increasing ability of physicians to identify women with elevated breast cancer risk, prophylactic methods are still currently limited to physical monitoring and prophylactic mastectomy.
In view of the above, what is needed in the art is the identification of novel methods which aid in the prevention and treatment of cancers of the mammary gland. In this context, optimal methods are those which have a wide application both in the diagnosis of cancer, as well as the prophylactic and therapeutic treatment of cancer.
The present invention is based on the observations of the respective roles of the epithelial and myoepithelial cell lineages in the development of breast cancer and the fact that these cell types differentially express gene products that can be targeted by cell specific vectors. In this context, new strategies emerge which can be used as a means of breast carcinoma diagnosis, prophylaxis and treatment. A first strategy is to selectively target cells of the breast epithelium so that breast carcinoma does not develop. A second strategy is to target myoepithelial cells as a means of bolstering the myoepithelial defense so that even if DCIS develops, it will be confined to the ductal system indefinitely.
The present invention provides prophylactic and therapeutic methods of treating a disease of the ductal epithelium of the mammary gland, in particular cancer. The present invention further provides diagnostic methods of assessing the status of cancers of the mammary gland. In this context, the present invention provides methods directed to selectively transducing a cell in a ductal system in a mammary gland comprising the step of using ductal cannulation to contact the cell with a vector that selectively targets that cell. In an illustrative embodiment, the invention consists of a method of selectively transducing either a epithelial cell or a myoepithelial cell, by contacting the cell with a vector that selectively targets a CAR molecule that is expressed on the epithelial cell or a heparin sulfate proteoglycan molecule that is expressed on the myoepithelial cell.
In a specific embodiment of the invention, the vector is a replication defective adenovirus which targets a molecule expressed solely by a epithelial cell and subsequently induces cell death by delivery of an cytolysis inducing gene such as thymidine kinase or cytosine deaminase. In a more preferred embodiment, the vector is a replication-competent lytic adenovirus which targets a coxsackievirus and adenovirus receptor (CAR) molecule expressed by the epithellal cell and subsequently induces cell death by lysis. In a highly preferred embodiment, the replication-competent lytic adenovirus contains a cis element such as a lactoalbumin promoter and the MUCI promoter which stimulates adenovirus replication in the presence of a trans factor present in the epithelial cell and induces more effective lysis. The myoepithelial cells of the breast duct, which lack CAR expression (as shown, for example by RT-PCR), are completely resistant to adenovirus infection (transduction) and serve as a barrier to systemic infection.
In another specific embodiment of the invention, the vector is a recombinant adeno-associated virus which targets a molecule expressed by a myoepithelial cell and comprises a polynucleotide which encodes a polypeptide which inhibits the development of epithelial cell cancer. In a specific embodiment, the polypeptide of the recombinant adeno-associated virus inhibits angiogenesis or the proliferation, invasion or metastases of a epithelial cell. In a specific embodiment, the polypeptide is maspin, thrombospondin-1, TIMP-1, protease nexin-II, xcex1-1 antitrypsin or soluble bFGF receptor. In a more preferred embodiment, the recombinant adeno-associated virus contains a cis element such as a lactoalbumin promoter and the MUCI promoter which stimulates recombinant adeno-associated virus replication in the presence of a trans factor present in the epithelial cell.
The present invention also provides a method of treating the ductal epithelium of a mammary gland prophylactically for cancer, which method comprises the step of contacting, by ductal cannulation, the ductal epithelium of the mammary gland with a tissue specific vector so as to inhibit the formation of cancer of ductal epithelial origin. In addition, the present invention provides combined therapeutic/prophylactic methods of treating the mammary gland therapeutically by surgery, radiation and/or chemotherapy and, either concomitantly or subsequently, contacting the ductal epithelium of the mammary gland with a cell specific vector which specifically targets a epithellal or a myoepithelial cell.
The present invention also provides a method of determining the lineage of a cell in a ductal system in a mammary gland selected from the group consisting of a luminal epithelial cell and a myoepithelial cell, by using ductal cannulation to contact the cell with vector containing a reporter gene, wherein the vector selectively targets a molecule that is expressed on the epithelial cell or the myoepithelial cell.
In addition, the present invention provides compositions including recombinant adenovirus and recombinant adeno-associated virus vectors as well as myoepithelial cell lines and transplantable xenografts.
FIG. 1 is a schematic which depicts the ductal-lobular unit of the breast and illustrates the possibility of gene therapy strategies targeting either epithelial cells and/or myoepithelial cells.
FIGS. 2A is a photograph illustrating the feasibility of gaining access to the entire ductal system of the breast through nipple duct identification and cannulation.
FIG. 2B, a photograph showing that when nipple ducts are individually cannulated, an injected dye reaches every ductal orifice.
FIG. 3A is a photograph showing the absence of xcex2-galactosidase expression in myoepithelial cells contacted with a xcex2-galactosidase containing recombinant adenovirus (Ad2) to illustrate how the expression of CAR on the surface of epithelial cells in the ductal-lobular unit of the breast allows the selective targeting of the epithelial cells and how the absence of CAR on myoepithelial cells confers resistance of this cell to adenoviral infection.
FIG. 3B is a photograph showing the intense xcex2-galactosidase expression in epithelial cells contacted with a xcex2-galactosidase containing recombinant adenovirus as an illustration of how the expression of CAR on the surface of epithelial cells in the ductal-lobular unit of the breast allows the selective targeting of epithelial cells.
FIG. 3C is a photograph showing how CAR expression is completely absent in myoepithelial cells (lane under CAR) but present in ductal epithelial cells and carcinoma lines (other lanes).
FIG. 4A is a photograph showing reporter gene expression in myoepithelial cells contacted with a recombinant adeno-associated virus containing a human green fluorescence reporter gene which illustrates how the expression of heparin sulfate proteoglycan on the surface of myoepithelial cells in the ductal-lobular unit of the breast allows the selective targeting of the myoepithelial cells.
FIG. 4B is a photograph showing only background reporter gene expression in epithelial cells contacted with a recombinant adeno-associated virus containing a human green fluorescence reporter gene which illustrates how the expression of heparin sulfate proteoglycan on the surface of myoepithelial cells in the ductal-lobular unit of the breast allows the selective targeting of the myoepithelial cells.
FIG. 5 is a Southern blot of DNA cut with Hinf I and probed using the multi-locus Jefferys probe (33.6) showing the distinct and identifying pattern of a number of illustrative myoepithelial cell lines and xenografts disclosed herein (HMS-1, HMS-X, HMS-3, HMS-3X, HMS-4X). All of the myoepithelial cell lines and xenografts exhibited the same susceptibility to recombinant adeno-virus associated virus transfection and the same resistance to adenoviral transfection. Other myoepithelial cell lines and xenografts (HMS-5X, HMS-6X, HMS-5, HMS-6) not depicted exhibit the same properties.
FIG. 6 is a Southern blot of DNA cut with Hae III and probed using the multi-locus Jefferys probe (33.6) showing the distinct and identifying pattern of a number of illustrative myoepithelial cell lines and xenografts disclosed herein (HMS-1, HMS-X, HMS-3, HMS-3X, HMS-4X). All of the myoepithelial cell lines and xenografts exhibited the same susceptibility to recombinant adeno-virus associated virus transfection and the same resistance to adenoviral transfection. Other myoepithelial cell lines and xenografts (HMS-5X, HMS-6X, HMS-5, HMS-6) not depicted exhibit the same properties.
FIG. 7 is a bar graph comparing MDA-MB-231 breast carcinoma cell invasion in MATRIGEL matrix in the presence of no myoepithelial cells (control), HMS myoepithelial cells (HMS), rAAV transfected HMS myoepithelial cells (rAAV-HMS) and rAAV-recombinant maspin transfected HMS myoepithelial cells (rAAV-maspin-HMS). The results demonstrate that myoepithelial cells which overexpress maspin are highly effective at blocking carcinoma cell invasion in MATRIGEL matrix. Specifically, the effects of transfected myoepithelial clones expressing recombinant maspin in invasion inhibition assays show a 200% increase in inhibition of invasion.